Methods for producing fusion polypeptides or enhancing expression of fusion polypeptides

ABSTRACT

The present invention relates to the cloning and expression of foreign protein or polypeptides in bacteria, such as  Escherichia coli . In particular, this invention relates to expression tools comprising a FKBP-type peptidyl prolyl isomerase selected from the group consisting of FkpA, SlyD, and trigger factor; methods of recombinant protein expression, the recombinant polypeptides thus obtained, as well as to the use of such polypeptides.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.10/179,038, filed Jun. 24, 2002, which claims priority to EP ApplicationNo. 01115225.3 filed on Jun. 22, 2001; and EP Application No. 01120939.2filed on Aug. 31, 2001, both in the European Patent Office, which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the cloning and expression of aheterologous protein or polypeptide in bacteria such as Escherichiacoli. In particular, this invention relates to expression toolscomprising a FKBP-type peptidyl prolyl isomerase selected from the groupconsisting of FkpA, SlyD, and trigger factor, methods of recombinantprotein expression, the recombinant polypeptides thus obtained, as wellas to the use of such polypeptides.

BACKGROUND

A large variety of expression systems has been described in the patentas well as in the scientific literature. However, despite the fact thatfusion proteins have become a cornerstone of modern biology, obtainingthe target protein in a soluble, biologically active form, as well as inhigh yield, continues to be a major challenge (Kapust, R. B. and Waugh,D. S., Protein Sci 8 (1999) 1668-74).

Examples of fusion partners that have been touted as solubilizing agentsinclude thioredoxin (TRX), glutathione S-transferase (GST),maltose-binding protein (MBP), Protein A, ubiquitin, and DsbA. Althoughwidely recognized and potentially of great importance, this solubilizingeffect remains poorly understood. It is not clear, for example, whatcharacteristics besides intrinsically high solubility epitomize aneffective solubilizing agent. Are all soluble fusion partners equallyproficient at this task, or are some consistently more effective thanothers? Similarly, it is not known whether the solubility of manydifferent polypeptides can be improved by fusing them to a highlysoluble partner, or whether this approach is only effective in a smallfraction of cases.

The state of the art relating to the most potent expression systems hasrecently been summarized by Kapust et al., supra. In their attempt toproduce soluble fusion proteins comprising various target proteins theyassessed three different and prominent candidate fusion partners.Maltose-binding protein (MBP), glutathione S-transferase (GST), andthioredoxin (TRX) were tested for their ability to inhibit theaggregation of six diverse proteins that normally accumulate in aninsoluble form. All these candidate expression systems are known to theskilled artisan and described in detail elsewhere (e.g., EP 293 249describes in detail the use of GST as an expression tool).

Remarkably, Kapust et al., supra, found that MBP is a far more effectivesolubilizing agent than the other two fusion partners also widely usedin the art. Moreover, they demonstrated that only in some cases, fusionto MBP can promote the proper folding of the attached protein into itsbiologically active conformation.

It is especially critical that many aggregation-prone polypeptides maybe rendered soluble by fusing them to an appropriate partner, but thatsome candidate fusion partners in a more or less unpredictable way aremuch better solubilizing agents than others.

A great demand therefore exists to provide for alternative, efficientexpression tools, which are especially appropriate for the recombinantexpression of aggregation prone proteins, e.g., like the rsgps.

There is a wealth of patent literature relating to proteins which bindto the immunosupressant FK-506, the so-called FK-506 binding proteins orFKBPs.

These proteins have been extensively studied and commercial applicationshave been designed centering around the FK-506 binding activity of theseproteins. For example, WO 93/25533 makes use ofCTP:CMP-3-deoxy-D-manno-octulosonate cytidyl transferase (=CKS) asexpression tool. A FKBP is inserted into a CKS-based expression vectordown-stream of the CKS gene. The fusion protein obtained is used toimprove measurements of FK-506 and other immunosuppressants.

WO 00/28011 discloses materials and methods for regulation of biologicalevents such as target gene transcription and growth, proliferation anddifferentiation of engineered cells.

WO 97/10253 relates to a high throughput assay for screening ofcompounds capable of binding to a fusion protein which consists of atarget protein and an FK-506-binding protein. Disclosed is the use of aFKBP12-Src homology (SH2) fusion protein in an high throughput screeningassay. The fusion protein is produced in soluble form in the bacterialperiplasm and released by standard freeze-thaw treatment.

SUMMARY OF THE INVENTION

The present invention in a first embodiment relates to a recombinant DNAmolecule, encoding a fusion protein, comprising at least one nucleotidesequence coding for a target polypeptide and upstream thereto at leastone nucleotide sequence coding for a FKBP chaperone, characterized inthat the FKBP chaperone is selected from the group consisting of FkpA,SlyD and trigger factor.

Preferred ways of designing such recombinant DNA molecules as well astheir use as part of an expression vector, a host cell comprising suchexpression vector, and in the production of fusion polypeptide are alsodisclosed.

It has in addition been found that the recombinant fusion polypeptidesthemselves exhibit surprising and advantageous properties, e.g., withregard to solubilization, purification and handling. In a furtherembodiment the present invention relates to a recombinantly-producedfusion protein comprising at least one polypeptide sequencecorresponding to a FKBP chaperone selected from the group consisting ofFkpA, SlyD and trigger factor and at least one polypeptide sequencecorresponding to a target peptide.

A further embodiment relates to a recombinantly-produced fusion proteincomprising at least one polypeptide sequence corresponding to a FKBPchaperone selected from the group consisting of FkpA, SlyD and triggerfactor, at least one polypeptide sequence corresponding to a targetpolypeptide, and at least one peptidic linker sequence of 10-100 aminoacids.

Preferred recombinant fusion polypeptides are also disclosed as well asthe use of such fusion polypeptides in various applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 UV spectrum of FkpA-gp41 at pH 2.5

UV-spectrum of the fusion polypeptide FkpA-Gp41 after dialysis against50 mM sodium phosphate, pH 2.5; 50 mM NaCl. Surprisingly, the two-domainconstruct remains completely soluble after removal of the solubilizingchaotropic agent GuHCl. There is no evidence for the existence oflight-straying aggregates that would be expected to cause a baselinedrift and significant apparent absorption at wavelengths beyond 300 nm.

FIG. 2 Near UV CD spectrum of FkpA-gp41 at pH 2.5

The spectrum was recorded on a Jasco 720 spectropolarimeter in 20 mMsodium phosphate, pH 2.5; 50 mM NaCl at 20° C. and was accumulated ninetimes to lower the noise. Protein concentration was 22.5 μM at a pathlength of 0.5 cm. The aromatic ellipticity shows the typical signatureof gp41. At pH 2.5, FkpA is largely unstructured and does not contributeto the signal in the Near-UV-CD at all.

FIG. 3 Far UV CD spectrum of FkpA-gp41 at pH 2.5

The spectrum was recorded on a Jasco 720 spectropolarimeter in 20 mMsodium phosphate pH 2.5; 50 mM NaCl at 20° C. and was accumulated ninetimes to improve the signal-to-noise ratio. Protein concentration was2.25 μM at a path-length of 0.2 cm. The minima at 220 and 208 nm pointto a largely helical structure of gp41 in the context of the fusionprotein. The spectural noise below 197 nm is due to the high amideabsorption and does not report on any structural features of the fusionprotein. Nevertheless, the typical helix-maximum at 193 nm can beguessed.

FIG. 4 Near UV CD of FkpA-gp41 under physiological buffer conditions.

The spectrum was recorded on a Jasco 720 spectropolarimeter in 20 mMsodium phosphate, pH 7.4; 50 mM NaCl at 20° C. and was accumulated ninetimes to lower the noise. Protein concentration was 15.5 μM at apath-length of 0.5 cm. Strikingly, the aromatic ellipticity of thecovalently linked protein domains of g41 and FkpA (continuous line) ismade up additively from the contributions of native-like all-helicalgp41 and pH 3.0 (lower dashed line) and the contributions of FkpA at pH7.4 (upper dashed line). This indicates that the carrier FkpA and thetarget gp41 (i.e. two distinct functional folding units) refoldreversibly and quasi-independently when linked in a polypeptide fusionprotein.

FIG. 5 Far UV CD of FkpA-gp41 under physiological buffer conditions.

The spectrum was recorded on a Jasco 720 spectropolarimeter in 20 mMSodium phosphate, pH 7.4; 50 mM NaCl at 20° C. and accumulated ninetimes to improve the signal-to-noise ratio. Protein concentration was1.55 μM at a path-length of 0.2 cm. The strong signals at 222 nm and 208nm, respectively, point to a largely helical structure of gp41 in thecontext of the fusion construct. The noise below 198 nm is due to thehigh protein absorption and does not reflect any secondary structuralproperties of FkpA-gp41.

FIG. 6 The Near-UV-CD-spectra of scFkpA and scSlyD resemble each other

CD spectra were recorded on a Jasco-720 spectropolarimeter in 0.5cm-cuvettes and averaged to improve the signal-to-noise-ratio. Bufferconditions were 50 mM sodium phosphate pH 7.8, 100 mM sodium chloride at20° C. Protein concentration was 45 μM for both scFkpA (top line at 280nm) and scSlyD (lower line at 280 nm), respectively. The structuralsimilarity of both proteins is evidenced by the similar signature in the“fingerprint region.”

DETAILED DESCRIPTION

The present invention describes novel polypeptide expression systems. Ina preferred embodiment it relates to a recombinant DNA molecule,encoding a fusion protein, comprising at least one nucleotide sequencecoding for a target polypeptide and upstream thereto at least onenucleotide sequence coding for a FKBP chaperone, characterized in thatthe FKBP chaperone is selected from the group consisting of FkpA, SlyDand trigger factor.

It was the task of the present invention to investigate whether it ispossible to develop and provide efficient alternative expression systemswhich can be used for improved expression of a recombinant proteincomprising a rsgp as a target protein and which at the same time arealso appropriate for less critical target proteins.

To our surprise we have been able to identify certain modular members ofthe FKBP-type family of the peptidyl prolyl isomerase (PPI or PPIase)chaperones as very promising cloning tools. We found that an expressionsystem based on a FKBP-type family of the chaperone selected from thegroup consisting of SlyD, FkpA, and trigger factor is ideal to expresscritical proteins like an rsgp and at the same time we could alsodemonstrate that these chaperones as well represent extremely promisingcloning tools for less critical target proteins.

As the skilled artisan will appreciate the term “at least one” is usedto indicate that one or more nucleotide sequences coding for a targetpolypeptide, or for a FKBP chaperone, respectively, may be used inconstruction of a recombinant DNA molecule without departing from thescope of the present invention. Preferably, the DNA construct willcomprise one or two sequences coding for a target polypeptide, one beingmost preferred, and at the same time, will contain at least one and atmost four sequences, coding for a chaperone, one or two being mostpreferred.

The term “recombinant DNA molecule” refers to a DNA molecule which ismade by the combination of two otherwise separated segments of sequenceaccomplished by the artificial manipulation of isolated segments ofpolynucleotides by genetic engineering techniques or by chemicalsynthesis. In so doing, one may join together polynucleotide segments ofdesired functions to generate a desired combination of functions.

Large amounts of the polynucleotides may be produced by replication in asuitable host cell. Natural or synthetic DNA fragments coding forproteins or fragments thereof will be incorporated into recombinantpolynucleotide constructs, typically DNA constructs, capable ofintroduction into a replication in a prokaryotic or eukaryotic cell. Thepolynucleotides may also be produced by chemical synthesis, including,but not limited to, the phosphoramidite method described by Beaucage, S.L. and Caruthers, M. H., Tetrahedron Letters 22 (1981) 1859-1862 and thetriester method according to Matteucci, M. D. and Caruthers, M. H., J.Am. Chem. Soc. 103 (1981) 3185-3191. A double-stranded fragment may beobtained from the single-stranded product of chemical synthesis eitherby synthesizing the complementary strand and annealing the strandstogether under appropriate conditions or by adding the complementarystrand using DNA polymerase with an appropriate primer sequence.

A polynucleotide is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods known in the art, thepolynucleotide can be transcribed and/or translated to produce thepolypeptide or a fragment thereof.

A target polypeptide according to the present invention may be anypolypeptide required in larger amounts and therefore difficult toisolate or purify from other non-recombinant sources. Examples of targetproteins preferably produced by the present methods include mammaliangene products, such as enzymes, cytokines, growth factors, hormones,vaccines, antibodies and the like. More particularly, preferredoverexpressed gene products of the present invention include geneproducts such as erythropoietin, insulin, somatotropin, growth hormonereleasing factor, platelet derived growth factor, epidermal growthfactor, transforming growth factor a, transforming growth factor 13,epidermal growth factor, fibroblast growth factor, nerve growth factor,insulin-like growth factor I, insulin-like growth factor II, clottingFactor VIII, superoxide dismutase, a - interferon, y-interferon,interleukin-1, interleukin-2, interleukin-3, interleukin-4,interleukin-5, interleukin-6, granulocyte colony stimulating factor,multi-lineage colony stimulating activity, granulocyte-macrophagestimulating factor, macrophage colony stimulating factor, T cell growthfactor, lymphotoxin and the like. Preferred overexpressed gene productsare human gene products. Moreover, the present methods can readily beadapted to enhance secretion of any overexpressed gene product which canbe used as a vaccine. Overexpressed gene products which can be used asvaccines include any structural, membrane-associated, membrane-bound orsecreted gene product of a mammalian pathogen. Mammalian pathogensinclude viruses, bacteria, single-celled or multi-celled parasites whichcan infect or attack a mammal. For example, viral vaccines can includevaccines against viruses such as human immunodeficiency virus (HIV),vaccinia, poliovirus, adenovirus, influenza, hepatitis A, hepatitis B,dengue virus, Japanese B encephalitis, Varicella zoster,cytomegalovirus, hepatitis A, rotavirus, as well as vaccines againstviral diseases like measles, yellow fever, mumps, rabies, herpes,influenza, parainfluenza and the like. Bacterial vaccines can includevaccines against bacteria such as Vibrio cholerae, Salmonella typhi,Bordetella pertussis, Streptococcus pneumoniae, Hemophilus influenza,Clostridium tetani, Corynebacterium diphtheriae, Mycobacterium leprae,R. rickettsii, Shigella, Neisseria gonorrhoeae, Neisseria meningitidis,Coccidioides immitis, Borellia burgdorferi, and the like.

Preferably, the target protein is a member of a group consisting ofHIV-1 gp41, HIV-2 gp36, HTLV gp21, HIV-1 p17, SlyD, FkpA, and triggerfactor.

A target polypeptide according to the present invention may alsocomprise sequences; e.g., diagnostically relevant epitopes, from severaldifferent proteins constructed to be expressed as a single recombinantpolypeptide.

The folding helpers termed peptidyl prolyl isomerases (PPIs or PPlases)are subdivided into three families, the parvulines (Schmid, F. X.,Molecular chaperones in the life cycle of proteins (1998) 361-389, Eds.A. L. Fink and Y. Goto, Marcel Decker In., New York), Rahfeld, J. U., etal., FEBS Lett 352 (1994) 180-4) the cyclophilines (Fischer, G., et al.,Nature 337 (1989) 476-8, and the FKBP family (Lane, W. S., et al., JProtein Chem 10 (1991) 151-60). The FKBP family exhibits an interestingbiochemical feature since its members have originally been identified bytheir ability to bind to macrolides, e.g., FK 506 and rapamycin (Kay, J.E., Biochem J. 314 (1996) 361-85).

According to the present invention, the preferred modular PPlases areFkpA (Ramm, K. and Pluckthun, A., J Biol Chem 275 (2000) 17106-13), SlyD(Hottenrott, S., et al., J Biol Chem 272 (1997) 15697-701) and triggerfactor (Scholz, C., et al., Embo J 16 (1997) 54-8), all members of theFKBP family. Most preferred are the chaperones FkpA and SlyD.

It is also well known and appreciated that it is not necessary to alwaysuse the complete sequence of a molecular chaperone. Functional fragmentsof chaperones (so-called modules), which still possess the requiredabilities and functions, may also be used (cf. WO 98/13496).

For instance, FkpA is a periplasmic PPI that is synthesized as aninactive precursor molecule in the bacterial cytosol and translocatedacross the cytoplasmic membrane. The active form of FkpA (mature FkpA orperiplasmic FkpA) lacks the signal sequence (amino acids 1 to 25) andthus comprises amino acids 26 to 270 of the precursor molecule. Relevantsequence information relating to FkpA can easily be obtained from publicdatabases, e.g., from “SWISS-PROT” under accession number P45523. TheFkpA used as expression tool according to the present invention lacksthe N-terminal signal sequence.

A close relative of FkpA, namely SlyD, consists of a structuredN-terminal domain responsible for catalytic and chaperone functions andof a largely unstructured C-terminus that is exceptionally rich inhistidine and cysteine residues (Hottenrott, supra). We found that aC-terminally truncated variant of SlyD comprising amino acids 1-165exerts exceptionally positive effects on the efficient expression oftarget proteins. Unlike in the wild-type SlyD, the danger ofcompromising disulfide shuffling is successfully circumvented in thetruncated SlyD-variant (1-165) used. A recombinant DNA moleculecomprising a truncated SlyD (1-165) represents a preferred embodiment ofthe present invention.

In a preferred mode of designing a DNA construct according to thepresent invention, no signal peptides are included. The expressionsystems according to the present invention have been found mostadvantageous when working as cytosolic expression system. This cytosolicexpression results in the formation of inclusion bodies. Different fromthe pronounced and well-known problems usually associated with inclusionbodies, we now have found that not only an exceptionally high amount ofprotein is produced, but that the recombinant proteins according to thepresent invention are also easy to handle, e.g., easy to solubilize andto refold. In a preferred embodiment the present invention thus relatesto a recombinant DNA molecule, encoding a fusion protein, comprising atleast one nucleotide sequence coding for a target polypeptide andupstream thereto at least one nucleotide sequence coding for a FKBPchaperone, wherein the FKBP chaperone is selected from the groupconsisting of FkpA, SlyD and trigger factor further characterized inthat the DNA construct lacks a signal peptide.

The term “lacks a signal peptide” must not be understood as an unduelimitation. As the skilled artisan will readily appreciate either theconstruct may in fact lack the signal peptide sequence. As analternative, however, the sequence may simply be modified to lack signalpeptide function.

Variants of the above-discussed chaperones, bearing one or several aminoacid substitutions or deletions, may also be used to obtain arecombinant DNA or a fusion polypeptide according to the presentinvention. The skilled artisan can easily assess whether such variants,e.g., fragments or mutants of chaperones or chaperones from alternativesources, are appropriate for a method of the invention by using theprocedures as described in the Examples section.

The term “recombinant” or “fusion polypeptide” as used in the presentinvention, refers to a polypeptide comprising at least one polypeptidedomain corresponding to the FKBP-chaperone used as expression tool andat least one polypeptide domain corresponding to the target protein.Optionally such fusion proteins may additionally comprise a linkerpolypeptide of 10-100 amino acid residues. As the skilled artisan willappreciate, such a linker polypeptide is designed as most appropriatefor the intended application, especially in terms of length,flexibility, charge, and hydrophilicity.

Preferably, the DNA construct of the present invention encodes a fusionprotein comprising a polypeptide linker in between the polypeptidesequence corresponding to the FKBP-chaperone and the polypeptidesequence corresponding to the target protein. Such a DNA sequence codingfor a linker, in addition to e.g., providing for a proteolytic cleavagesite, may also serve as a polylinker, i.e., it may provide multiple DNArestriction sites to facilitate fusion of the DNA fragments coding for atarget protein and a chaperone domain.

The present invention makes use of recombinant DNA technology in orderto construct appropriate DNA molecules.

In a further preferred embodiment, the present invention relates arecombinant DNA molecule, encoding a fusion protein, comprisingoperably-linked at least one nucleotide sequence coding for a targetpolypeptide and upstream thereto at least one nucleotide sequence codingfor a FKBP chaperone, characterized in that the FKBP chaperone isselected from the group consisting of FkpA, SlyD and trigger factor.

Polynucleotide sequences are operably-linked when they are placed into afunctional relationship with another polynucleotide sequence. Forinstance, a promoter is operably-linked to a coding sequence if thepromoter affects transcription or expression of the coding sequence.Generally, operably-linked means that the linked sequences arecontiguous and, where necessary to join two protein coding regions, bothcontiguous and in reading frame. However, it is well known that certaingenetic elements, such as enhancers, may be operably-linked even at adistance, i.e., even if not contiguous.

As the skilled artisan will appreciate it is often advantageous todesign a nucleotide sequence coding for a fusion protein such that oneor a few, e.g., up to nine, amino acids are located in between the twopolypeptide domains of said fusion protein. Fusion proteins thusconstructed, as well as the DNA molecules encoding them, obviously arealso within the scope of the present invention.

DNA constructs prepared for introduction into a host typically comprisea replication system recognized by the host, including the intended DNAfragment encoding the desired target fusion peptide, and will preferablyalso include transcription and translational initiation regulatorysequences operably-linked to the polypeptide encoding segment.Expression systems (expression vectors) may include, for example, anorigin of replication or autonomously replicating sequence (ARS) andexpression control sequences, a promoter, an enhancer and necessaryprocessing information sites, such as ribosome-binding sites, RNA splicesites, polyadenylation sites, transcriptional terminator sequences, andmRNA stabilizing sequences.

The appropriate promoter and other necessary vector sequences areselected so as to be functional in the host. Examples of workablecombinations of cell lines and expression vectors include but are notlimited to those described Sambrook, J., et al., in “Molecular Cloning:A Laboratory Manual” (1989)-, Eds. J. Sambrook, E. F. Fritsch and T.Maniatis, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, orAusubel, F., et al., in “Current Protocols in Molecular Biology” (1987and periodic updates), Eds. F. Ausubel, R. Brent and K. R. E., Wiley &Sons Verlag, New York; and Metzger, D., et al., Nature 334 (1988) 31-6.Many useful vectors for expression in bacteria, yeast, mammalian,insect, plant or other cells are known in the art and may be obtainedfrom vendors including, but not limited to, Stratagene, New EnglandBiolabs, Promega Biotech, and others. In addition, the construct may bejoined to an amplifiable gene (e.g., DHFE) so that multiple copies ofthe gene may be obtained.

Expression and cloning vectors will likely contain a selectable marker,a gene encoding a protein necessary for the survival or growth of a hostcell transformed with the vector, although such a marker gene may becarried on another polynucleotide sequence co-introduced into the hostcell. Only those host cells expressing the marker gene will surviveand/or grow under selective conditions. Typical selection genes includebut are not limited to those encoding proteins that (a) conferresistance to antibiotics or other toxic substances, e.g., ampicillin,tetracycline, etc.; (b) complement auxotrophic deficiencies; or (c)supply critical nutrients not available from complex media. The choiceof the proper selectable marker will depend on the host cell, andappropriate markers for different hosts are known in the art.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any method known in the art. These methods varydepending upon the type of cellular host, including but not limited totransfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, other substances, and infection by viruses.Large quantities of the polynucleotides and polypeptides of the presentinvention may be prepared by expressing the polynucleotides of thepresent invention in vectors or other expression vehicles in compatiblehost cells. The most commonly used prokaryotic hosts are strains ofEscherichia coli, although other prokaryotes, such as Bacillus subtilismay also be used. Expression in Escherichia coli represents a preferredmode of carrying out the present invention.

Construction of a vector according to the present invention employsconventional ligation techniques. Isolated plasmids or DNA fragments arecleaved, tailored, and religated in the form desired to generate theplasmids required. If desired, analysis to confirm correct sequences inthe constructed plasmids is performed in a known fashion. Suitablemethods for constructions expression vectors, preparing in vitrotranscripts, introducing DNA into host cells, and performing analysesfor assessing expression and function are known to those skilled in theart. Gene presence, amplification and/or expression may be measured in asample directly, for example, by conventional Southern blotting,Northern blotting to quantitate the transcription of mRNA, dot blotting(DNA or RNA analysis), or in situ hybridization, using an appropriatelylabeled probe which may be based on a sequence provided herein. Thoseskilled in the art will readily envisage how these methods may bemodified, if desired.

In a preferred embodiment, a recombinant DNA molecule according to thepresent invention comprises a single nucleotide sequence coding for aFKBP-chaperone selected from the group consisting of FkpA, SlyD, andtrigger factor and a single nucleotide sequence coding for a targetpolypeptide.

A fusion protein comprising two FKBP-chaperone domains and one targetprotein domain is also very advantageous. In a further preferredembodiment the recombinant DNA molecule according to the presentinvention comprises two sequences coding for a FKBP-chaperone and onesequence coding for a target polypeptide.

The DNA molecule may be designed to comprise both the DNA sequencescoding for the FKBP-chaperone upstream to the target protein.Alternatively the two FKBP-domains may be arranged to sandwich thetarget protein. The construct comprising both FKBP-domains upstream tothe target protein represents a preferred embodiment according to thepresent invention.

The DNA construct comprising two chaperone domains as well as a targetpolypeptide domain preferably also contains two linker peptides inbetween these domains. In order to allow for systematic cloning, thenucleotide sequences coding for these two linker peptide sequencespreferably are different. This difference in nucleotide sequence mustnot necessarily result in a difference in the amino-acid sequence of thelinker peptides. In yet a further preferred embodiment the amino acidsequences of the two linker peptides are identical. Such identicallinker peptide sequences for example are advantageous if the fusionprotein comprising two FKBP-chaperone domains as well as their targetprotein domain is to be used in an immunoassay.

In cases where it is desired to release one or all of the chaperones outof a fusion protein, according to the present invention, the linkerpeptide is constructed to comprise a proteolytic cleavage site. Arecombinant DNA molecule encoding a fusion protein comprising at leastone polypeptide sequence coding for a target polypeptide, upstreamthereto at least one nucleotide sequence coding for a FKBP-chaperoneselected from the group consisting of FkpA, SlyD, and trigger factor,and additionally comprising a nucleic acid sequence coding for apeptidic linker comprising a proteolytic cleavage site, represents afurther embodiment of this invention.

An expression vector comprising operably-linked to a recombinant DNAmolecule according to the present invention, i.e., a recombinant DNAmolecule encoding a fusion protein comprising at least onepolynucleotide sequence coding for a target polypeptide and upstreamthereto at least one nucleotide sequence coding for a FKBP-chaperone,wherein the FKBP-chaperone is selected from FkpA, SlyD, and triggerfactor, has proven to be very advantageous.

The expression vector comprising a recombinant DNA according to thepresent invention may be used to express the fusion protein in acell-free translation system or may be used to transform a host cell. Ina preferred embodiment, the present invention relates to a host celltransformed with an expression vector according to the presentinvention.

In a further preferred embodiment, the present invention relates to amethod of producing a fusion protein. Said method comprises the steps ofculturing a host cell transformed with an expression vector according tothe present invention, expression of that fusion protein in therespective host cell and purification of said fusion protein.

As discussed above, the FKBP-chaperone domain of FkpA, SlyD, or triggerfactor, respectively, is naturally or artificially constructed to yielda cytosolic fusion polypeptide expression. The fusion protein thusproduced is obtained in form of inclusion bodies. Whereas in the art,tremendous efforts are spent to obtain any desired recombinant proteinor the fusion protein directly in a soluble form, we have found that thefusion protein according to the present invention is easily obtained insoluble form from inclusion bodies. In a further preferred embodimentthe present invention therefore relates to a method of producing afusion protein according to the steps described above, wherein saidfusion protein is purified from inclusion bodies.

The purification of fusion protein from inclusion bodies is easilyachieved and performed according to standard procedures known to theskilled artisan, like chaotropic solubilization and various ways ofrefolding.

Isolation and purification of the fusion protein starts fromsolubilizing buffer conditions, i.e. from a buffer wherein the inclusionbodies, i.e., the fusion protein, are/is solubilized. An appropriatebuffer, which may be termed “non-physiological” or “solubilizing” bufferhas to meet the requirement that both the target protein and the FKBPchaperone are not irreversibly denatured. Starting from such bufferconditions, the chaperone is in close proximity to the target protein,and a change of the buffer conditions from non-physiological tophysiological conditions is possible without precipitation of the fusionprotein.

An appropriate (non-physiological) buffer, i.e., a buffer wherein boththe target protein which is essentially insoluble and the PPI-chaperoneare soluble either makes use of high or low pH, or of a high chaotropicsalt concentration or of a combination thereof. The solubilizing bufferpreferably is a buffer with rather a high concentration of a chaotropicsalt, e.g., 6.0 M guanidinium chloride at a pH of about 6. Uponrenaturation, both the target protein as well as the chaperone assumetheir native-like structure, and the chaperone exerts its positivesolubilizing effect.

In the context of this invention, physiological buffer conditions aredefined by a pH value between 5.0 and 8.5, and a total saltconcentration below 500 mM, irrespective of other non-salt ingredientsthat optionally may be present in the buffer (e.g., sugars, alcohols,detergents) as long as such additives do not impair the solubility ofthe fusion protein comprising the target protein and the chaperone.

A variety of target proteins has been expressed in large amounts.

The expression system, according to the present invention, for example,has been shown to work extremely well with biochemically ratherdifferent target proteins, e.g., SlyD, FkpA (proteins which are readilysoluble), HIV-1 p17 (a protein which is difficult to express in highamounts using conventional expression systems), HTLV gp 21 (a proteinwhich tends to aggregate), and HIV-1 gp41, as well as HIV-2 gp36 (bothproteins are extremely prone to aggregation and essentially insolubleunder physiological buffer conditions). As can be easily gathered fromExample 4 specifically relating to these proteins, the efficientexpression systems, according to the present invention, work and resultin high levels of fusion protein produced. Similar positive findingshave been made with a variety of other target proteins expressed asfusion protein, according to the present invention.

From the list of positive examples, it becomes readily obvious that thenovel expression system as disclosed in the present invention, providefor extremely attractive universal expression systems.

The expression systems, as disclosed herein, also have been compared tostandard expression systems making use of carrier proteins asrecommended in the art, like MBP. It has been found that the novelsystems with the target polypeptides tested are quite advantageous. Therelative yield of fusion protein produced according to the presentinvention was at least as good, and in the majority of cases, evenhigher as compared to the relative yield using MBP-based expression.Efficacy of expression can be assessed both in terms of yield of fusionprotein, e.g., per gram of E. coli cell mass or on a molar basis,comparing the concentrations of a target protein comprised in differentfusion proteins.

The present invention, in a preferred embodiment, relates to arecombinantly-produced fusion protein, comprising at least onepolypeptide sequence corresponding to a FKBP chaperone selected from thegroup consisting of FkpA, SlyD and trigger factor, and at least onepolypeptide sequence corresponding to a target peptide.

It has been found that the fusion proteins according to the presentinvention exhibit advantageous properties, thus e.g., facilitatingproduction, handling and use of otherwise critical proteins. Thisbecomes readily obvious from the description of the positive resultsobtained with a fusion protein comprising HIV-1 gp41. Whereasrecombinantly-produced gp41 itself is essentially insoluble, it isreadily soluble if present as part of a fusion protein according to thepresent invention.

In general a protein is considered “essentially insoluble” if in abuffer consisting of 20 mM sodium phosphate pH 7.4, 150 mM NaCl, it issoluble in a concentration of 50 nM or less. A fusion protein accordingto the present invention, comprising a FKBP chaperone and a targetprotein is considered “soluble” if, under physiological bufferconditions, e.g., in a buffer consisting of 20 mM sodium phosphate pH7.4, 150 mM NaCl, the target protein comprised in the PPI-chaperonecomplex is soluble in a concentration of 100 nM or more.

We found that the recombinantly-produced fusion protein, according tothe present invention, can be readily obtained from inclusion bodies insoluble form, even if the target protein is an aggregation-proneprotein, like HIV-1 gp41. A striking feature of gp41 comprised in arecombinantly-produced FkpA-gp41 is its exceptional solubility atphysiological buffer conditions as compared to the “unchaperoned” gp41ectodomain.

Moreover, it has been possible to demonstrate that the target proteincomprised in a fusion protein, according to the present invention,readily can be obtained in a native-like structure. Such native-likestructure, e.g., for HIV-1 gp41 has been confirmed by Near-UV-CD or byits immunoreactivity. Near-UV-CD analysis has shown the typical“gp41-signature,” which is known to the skilled artisan.

The fusion protein according to the present invention also is very easyto handle, e.g., it is quite easy to renature such fusion protein. It isinteresting that the “chaotropic material” (i.e. FkpA-gp41 in 6.0-7.0 MGuHCl) can be refolded in different ways, all resulting in athermodynamically stable and soluble native-like form. Refolding isachieved at high yields, both by dialysis and b rapid dilution, as wellas by renaturing size exclusion chromatography or matrix-assistedrefolding. These findings suggest that in this covalently linked form,the gp41-FkpA fusion polypeptide is a thermodynamically stable ratherthan a metastable protein.

Some of the FKBP-chaperones (e.g., FkpA) exert their chaperone functionin form of oligomers, i.e., in a complex comprising two or morenoncovalently associated FKBP polypeptides. We have surprisingly foundthat it is possible to design and produce such an active FKBP-dimer as asingle fusion protein on one and the same polypeptide. We have termedthese constructs single-chain PPIs, or single chain FKBPs. Thesingle-chain PPI comprising two SlyD domains therefore is termed scSlyDand the single-chain PPI comprising two FkpA domains therefore is termedscFkpA. A single-chain peptidyl-prolyl-isomerase, i.e. a fusion proteincomprising two PPI-domains represents a very advantageous and thereforepreferred embodiment of the present invention. The sc-PPI according tothe present invention may be a parvuline, a cyclophyline or a FKBP. Thesc-PPIs selected from the FKBP family of chaperones are preferred. Mostpreferred are sc SlyD and Sc FkpA, respectively.

A recombinantly-produced fusion protein comprising at least onepolypeptide sequence corresondence to a FKBP chaperone selected from thegroup consisting of FkpA, SlyD and trigger factor, at least onepolypeptide sequence corresponding to a target polypeptide, and at leastone peptidic linker sequence of 10-100 amino acids represents a furtherpreferred embodiment of the present invention.

As the skilled artisan will appreciate, the peptidic linker may beconstructed to contain the amino acids which are most appropriate forthe required application. E.g., in case of a hydrophobic target proteinthe linker polypeptide preferably will contain an appropriate number ofhydrophilic amino acids. the present invention specifically also relatesto fusion proteins which comprise the target polypeptide and one, or twoFKBP-chaperones or chaperone domains and an appropriate peptidic linkersequences between domains. For such applications where the targetprotein is required in free form a linker peptide or linker peptides areused, which contain an appropriate proteolytic cleavage site. Peptidesequences appropriate for proteolytic cleavage are well-known to theskilled artisan and comprise amongst others, e.g., Ile-Glu-Gly-Arg, SEQID NO:9, cleaved at the carboxy side of the arginine residue bycoagulation factor Xa, or Gly-Leu- Pro-Arg-Gly-Ser (SEQ ID NO:10), athrombin cleavage site, etc.

As mentioned above the fusion proteins according to the presentinvention can easily be obtained from inclusion bodies following asimple refolding scheme. They are readily soluble and targetpolypeptides comprised in such fusion proteins can easily be obtained innative-like confirmation. This is quite advantageous for polypeptidesderived from an infectious organism because such native-likepolypeptides are most advantageous in diagnostic as well as intherapeutic applications. In a preferred embodiment, the fusion protein,according to the present invention, is further characterized in that atarget protein is a polypeptide of interest as known from an infectiousorganism. Preferred infectious organisms, according to the presentinvention, are HIV, HTLV, and HCV.

From the scientific as well from the patent literature it is well-knownwhich peptide sequences contain diagnostically relevant epitopes. Forthe skilled artisan it is nowadays no problem to identify such relevantepitopes. In a further preferred embodiment the target proteincorresponding to a polypeptide derived from an infectious organism willcontain at least one diagnostically relevant epitope.

Due to their advantageous properties the recombinantly-produced fusionproteins according to the present invention in further preferredembodiments are used for the immunizations of laboratory animals, in theproduction of a vaccine or in an immunoassay, respectively.

In case a therapeutic application of the novel fusion proteins isintended, preferably a composition comprising a recombinantly-producedfusion protein according to the present invention and a pharmaceuticallyacceptable excipient are formulated.

The following examples, references, sequence listing and figures areprovided to aid the understanding of the present invention, the truescope of which is set forth in the appended claims. It is understoodthat modifications can be made in the procedures set forth withoutdeparting from the spirit of the invention.

EXAMPLES Example 1 Recombinant Production of HIV-1 gp41 using aFkpA-based Expression System

1.1 Construction of an Expression Plasmid Comprising FkpA and gp41

Wild-type FkpA was cloned, expressed and purified according to Bothmannand Plückthun, J Biol Chem 275 (2000) 17106-17113 with some minormodifications. For storage, the protein solution was dialyzed against 20mM NaH2PO4/NaOH (pH 6.0), 100 mM NaCl and concentrated to 26 mg/ml (1mM).

For cytosolic expression, the FkpA-coding sequence of the aboveexpression vector was modified to lack the sequence part coding for thesignal peptide and to comprise instead only the coding region of matureFkpA.

In the first step, the restriction site BamHI in the coding region ofthe mature E. coli FkpA was deleted using the QuikChange site-directedmutagenesis kit of Stratagene (La Jolla, Calif.; USA) with the primers:

5′ -gcgggtgttccgggtatcccaccgaattc-3′ (SEQ ID NO:1)5′ -gaattcggtgggatacccggaacacccgc-3′ (SEQ ID NO:2)The construct was named EcFkpA (ΔBamHl)[GGGS]₃.

HIV-1 gp41 (535-681)-His₆ was cloned and expressed in a T7promotor-based expression system. The gene fragment encoding amino acids535-681 from HIV-1 envelope protein was amplified by PCR from theT7-based expression vector using the primers:

(SEQ ID NO:3) 5′ -cgggatccggtggcggttcaggcggtggctctggtggcggtacgctgacggtacaggccag-3′ (SEQ ID NO:4) 5′ -ccgctcgaggtaccacagccaatttgttat-3′

The fragment was inserted into EcFkpA(A BamHI)[GGGS]₃ using BamHI andXhoI restriction sites.

The codons for a glycine-serine-rich linker [GGGS]₃ between FkpA ande-gp41 were inserted with reverse primer for cloning of FkpA and withforward primer for cloning of e-gp41.

The resulting construct was sequenced and found to encode the desiredprotein. Variants of this protein have also been generated bysite-directed mutagenesis according to standard procedures. A variant ofgp41 comprising four amino acid substitutions as compared to thewild-type sequence is, e.g., encoded by the DNA-constructs of SEQ IDNO:5 and 6, making use of FkpA or SlyD as expression system,respectively.

1.2 Purification of the FkpA-gp41 Fusion Protein from E. coli Cells

E. coli BL21 cells harboring the expression plasmid were grown to anOD₆₀₀ of 0.7, and cytosolic overexpression was induced by adding 1 mM ofIPTG at a growth temperature of 37° C. Four hours after induction, thecells were harvested by centrifugation (20 min at 5000 g). The bacterialpellet was resuspended in 50 mM sodium phosphate pH 7.8, 6.0 M GuHCl(guanidinium chloride), 5 mM imidazole and stirred at room temperature(10 min) for complete lysis. After repeated centrifugation (SorvallSS34,20000 rpm, 4° C.), the supernatant was filtered (0.8/0.2 μm) andapplied to a Ni-NTA-column (NTA: Nitrilotriacetate; Qiagen; Germantown,Md.), pre-equilibrated in lysis buffer. Unspecifically bound proteinswere removed in a washing step by applying 10 column volumes of lysisbuffer. Finally, the bound target protein was eluted with 50 mM sodiumphosphate, pH 2.5, 6.0 M GuHCl and was collected in 4 ml fractions. Theabsorbance was recorded at 280 nm.

The resulting acidic and chaotropic solution may be stored at 4° C. forfurther purification steps or in vitro refolding experiments.

Starting with this unfolding material, different refolding methods, suchas dialysis, rapid dilution, renaturing size exclusion chromatography ormatrix-assisted refolding can be used and carried out successfully, allof them leading to virtually the same native-like folded and solubleprotein.

1.3 Renaturation by Dialysis and Rapid Dilution

Material, solubilized as described above, is transferred intophysiological buffer conditions by dialysis. The chosen cut-off value ofthe dialysis tubing was 4000-6000 Daltons.

To induce refolding of the ectodomain (the HIV-1 gp41 part of the fusionprotein), GuHCl was removed from the eluted protein by dialysis against50 mM sodium phosphate, pH 2.5, 50 mM NaCl (sodium chloride). It is wellknown that the isolated ectodomain is all-helical and forms tertiarycontacts at this extreme pH. When analyzing recombinantly-produced FkpAby means of near UV CD, it was found that FkpA is essentiallyunstructured under the same conditions. It is surprising that refoldingof gp41-FkpA by dialysis results in a readily soluble protein complexcomprising the covalently linked gp41 and FkpA protein domains. The UVspectrum (FIG. 1) lacks stray light, i.e., apparent absorption beyond300 nm. Stray light would be indicative of aggregates, thus the spectrumshown in FIG. 1 implies that the re-folded material does not containsignificant amounts of aggregates.

Circular dichroism spectroscopy (CD) is the method of choice to assessboth secondary and tertiary structure of proteins. Ellipticity in thearomatic region (260-320 nm) reports on tertiary contacts within aprotein (i.e., the globular structure of a regularly folded protein),whereas ellipticity in the amide region reflects regular repetitiveelements in the protein backbone, i.e., secondary structure.

The near UV CD spectrum shown in FIG. 2 provides compelling evidencethat the ectodomain (in the context of the fusion protein) displaysnative-like tertiary contacts at pH 2.5. The spectrum of the covalentlylinked gp41/FkpA protein domains almost coincides with the spectrum ofthe isolated ectodomain under identical conditions (data not shown). Thetypical signature of gp41 was found: a maximum of ellipticity at 290 nm,a characteristic shoulder at 285 nm and another maximum at 260 nmreflecting an optically active disulfide bridge. it is important to notethat FkpA does not contribute to the near UV signal at all under therespective conditions. In fact, the aromatic ellipticity of FkpA at pH2.5 virtually equals the baseline (data not shown).

In agreement with the results from the near UV region, the far UV CD ofthe fusion construct at pH 2.5 points to a largely structured gp41molecule. The two maxima at 220 nm and 208 nm make up, and correspondto, the typical signature of an all-helical ectodomain (FIG. 3). Fromthe conditions indicated (50 mM sodium phosphate, pH 2.5, 50 mM NaCl),the FkpA-gp41 fusion polypeptide can easily be transferred tophysiological buffer conditions by rapid dilution. In conclusion, bothnear and far UV CD underline that native-like structured gp41 isavailable (in the context of the fusion protein also containing FkpA) ina very convenient fashion.

1.4 Renaturation by Size Exclusion Chromatography (SEC)

Unfolded gp41-FkpA polypeptide (dissolved in 50 mM sodium phosphate, pH7.8, 7.0 M GuHCl) was applied onto a Superdex 200 gel filtration columnequilibrated with 20 mM sodium phosphate, pH 7.4, 50 mM NaCl, 1 mM EDTA.FkpA-gp41 elutes essentially in three main fractions: as a highmolecular associate, as an apparent hexamer species and as an apparenttrimer species. The apparent trimer fraction was concentrated andassessed for its tertiary structure in a near UV CD measurement (FIG.4).

The resulting graph is virtually an overlay curve to which both thecarrier protein FkpA and the target protein gp41 contribute in a 1:1ratio. Most fortunately, gp41 displays tertiary structure at neutral pHand is evidently solubilized by the covalently bound chaperone. In otherwords, the chaperone FkpA seems to accept the native-like structuredectodomain gp41 as a substrate and to solubilize this hard-to-foldprotein at a neutral working pH. Thus, a crucial requirement forproducing high amounts of soluble gp41 antigen for diagnostic purposesis fulfilled.

The far UV CD of FkpA-gp41 at pH 7.4 (FIG. 5) confirms the near UV CDresults in that it shows the additivity of the signal contributions ofFkpA and gp41, respectively. As expected, the spectrum is dominated bythe highly helical gp41 ectodomain (maximal ellipticity at 220 nm and208 nm, respectively).

The data obtained with the covalently linked gp41/FkpA protein domainssolubilized at pH 7.4 under the conditions mentioned above indicate thatFkpA and gp41 behave as independently folding units within thepolypeptide construct.

Example 2 Use of a SlyD-Based Expression Vector

The chapereone SlyD has been isolated by routine cloning procedures fromE. coli. For recombinant expression, a DNA construct has been preparedcoding for amino acids 1 to 165 of SlyD. An expression vector has beenconstructed comprising SlyD(1-165) as fusion partner and HIV-1 gp41 astarget protein (cf: SEQ ID NO:6). The fusion protein was expressed andsuccessfully purified as described for FkpA-gp41 above. Interestingly,we found that a native-like fusion polypeptide of the SlyD (1-165)-gp41type can be obtained in a very convenient manner by dialysis of thechaotropic material (dissolved, e.g. in 7.0 M GuHCl) against 50 mMsodium phosphate pH 7.4, 150 mM NaCl at room temperature.

Example 3 Purification of scFkpA and scSlyD

The single-chain PPIases scSlyD (SEQ ID NO:7) and scFkpA (SEQ ID NO:8),respectively, were obtained from an E. coli overproducer according tovirtually the same purification protocol as described in Example 1. Inshort: the induced cells were harvested, washed in PBS and lysed in 50mM sodium phosphate pH 7.8, 100 mM sodium chloride, 7.0 M GuHCl at roomtemperature. The unfolded target proteins were bound to a Ni-NTA-columnvia their C-teminal hexa-His-tag and were refolded in 50 mM sodiumphosphate, pH 7.8, 100 mM sodium chloride. After this matrix-assistedrefolding procedure, the proteins were eluted in an imidazole gradientand subjected to a gel filtration on a Superdex 200® column.

Alternatively, scSlyD and scFkpA may be dialysed after elution to removeresidual concentrations of imidazole. Both proteins turn out to behighly soluble. ScSlyD, for example, does not tend to aggregate atconcentrations up to 25 mg/ml. In order to elucidate the tertiarystructure of the refolded scPPlases, we monitored CD-spectra in theNear-UV-region. The signatures of both scSlyD and scFkpA resemble eachother and reflect the close relationship and thus structural homology ofthe two FKBPs. Due to the low content in aromatic residues, the signalintensity of scSlyD (FIG. 6) is, however, significantly lower than theone of the scFkpA.

Example 4 Improved Expression of Target Proteins

The biochemically quite different target proteins HIV-1 gp41, HIV-2gp36, HIV-1 p17 and HTLV gp21 have been expressed using the pET/BL21expression system either without fusion partner (gp41, gp36, p17, gp21),or using same standard expression system, but comprising a DNA-constructcoding for a fusion protein according to the present invention(SlyD-gp41, FkpA-gp41, FkpA-p17, SlyD-gp36, FkpA-gp21). The efficiencyof these systems has been compared in terms of yield of recombinantprotein per E. coli cell mass [mg/g]. As becomes readily obvious fromTable 1, the novel expression systems lead to a significant improvementfor all proteins tested.

TABLE 1 Yield Protein [mg protein/g E. coli cell mass] gp41   ~1-2SlyD-gp41 ~30 FkpA-gp41 ~25 p17 ~1  FkpA-p17 ~15 gp36   ~1-2 SlyD-gp36~45 gp21 ~4  FkpA-gp21 ~30

References

Sambrook, J., et al., in “Molecular Cloning: A Laboratory Manual”(1989)-, Eds. J. Sambrook, E. F. Fritsch and T. Maniatis, Cold SpringHarbour Laboratory Press, Cold Spring Harbour, N.Y.

Beaucage, S. L. and Caruthers, M. H., Tetrahedron Letters 22 (1981)1859-1862

Ausubel, Fl, et al., in “Current protocols in molecular biology” (1987and periodic updates), Eds. F. Ausubel, R. Brent and K. R. E., Wiley &Sons Verlag, New York

Fischer, G., et al., Nature 337 (1989)476-8

Hottenrott, S., et al., J Biol Chem 272 (1997) 45697-701

Kapust, R. B. and Waugh, D. S., Protein Sci 8 (1999) 1668-74

Kay, J. E., Biochem J 314 (1996) 361-85

Lane, W. S., et al., Nature 334 (1988) 31-6

Matteucci, M. D. and Caruthers, M. H., J. Am. Chem. Soc. 103 (1981)3185-3191

Metzger, D., et al., Nature 334 (1988) 31-6

Rahfeld, J. U., et al. FEBS Lett 352 (1994) 180-4

Ramm, K. and Pluckthun, A., J Biol Chem 275 (2000) 17106-13

Schmid, F. X., Molecular chaperones in the life cycle of proteins (1998)361-389, Eds.

A. L. Fink and Y. Goto, Marcel Decker In., New York

Scholz, C., et al., Embo J 16 (1997) 54-8

EP 293 249

WO 00/28011

WO 93/25533

WO 97/10253

WO 98/1349

1-26. (canceled)
 27. A method of producing a fusion polypeptide,comprising: culturing a host cell comprising an expression vectorcomprising a recombinant DNA molecule encoding the fusion polypeptide,the recombinant DNA molecule comprising a first nucleotide sequencecoding for a target polypeptide and upstream to the first nucleotidesequence a second nucleotide sequence coding for an FKBP chaperone,wherein the FKBP chaperone provides for a functional chaperone activity,and wherein the FKBP chaperone allows for a cytosolic expression and invitro refolding of the fusion polypeptide; expressing the fusionpolypeptide; obtaining the fusion polypeptide in the cytosol;solubilizing the fusion polypeptide; and purifying the fusionpolypeptide.
 28. The method of claim 27, wherein the fusion polypeptideobtained in the cytosol is solubilized in a non-physiological buffer.29. The method of claim 28, wherein solubilizing is by chaotropicsolubilization.
 30. The method of claim 29, wherein thenon-physiological buffer comprises 6.0 M guanidinium chloride.
 31. Themethod of claim 27, further comprising refolding the fusion polypeptide.32. The method of claim 31, wherein refolding is by dialysis, rapiddilution, renaturing size exclusion chromatography, or matrix-assistedrefolding.
 33. The method of claim 27, comprising transferring thefusion polypeptide to physiological buffer conditions.
 34. The method ofclaim 33, wherein the physiological buffer conditions comprise a pH fromabout 5.0 to about 8.5 and a total salt concentration of less than 500mM.
 35. The method of claim 27, comprising confirming structure of thetarget polypeptide by Near-UV-CD analysis or circular dichroismspectroscopy.
 36. The method of claim 27, wherein the FKBP chaperone isselected from the group consisting of FkpA, SlyD and trigger factor. 37.The method of claim 27, wherein the FKBP chaperone comprises aC-terminally truncated wild-type SlyD chaperone.
 38. The method of claim38, wherein the C-terminally truncated wild- type SlyD chaperonecomprises amino acids 1-165 of the wild-type SlyD.
 39. The method ofclaim 27, wherein the FkpA chaperone comprises amino acids 26-270 of awild-type FkpA chaperone.
 40. The method of claim 27, wherein the hostcell is Escherichia coli.
 41. The method of claim 27, wherein therecombinant DNA molecule comprises a third nucleotide sequence codingfor a peptide linker of 10-100 amino acids located between the firstnucleotide sequence and the second nucleotide sequence.
 42. The methodof claim 27, wherein the target polypeptide is selected from the groupconsisting of HIV-1 gp41, HIV-2 gp36, HTLV gp21, and HIV-1 p17.
 43. Themethod of claim 27, wherein the target polypeptide comprises apolypeptide from an infectious organism.
 44. The method of claim 43,wherein the target polypeptide comprises a polypeptide comprising atleast one diagnostically relevant epitope of the infectious organism.45. A method for immunization of an experimental animal comprisingadministering the fusion polypeptide produced according to the method ofclaim 27 to the experimental animal.
 46. A vaccine comprising the fusionpolypeptide produced according to the method of claim
 27. 47. A methodfor increasing expression of a fusion polypeptide, comprising: culturinga host cell comprising an expression vector comprising a recombinant DNAmolecule encoding the fusion polypeptide, the recombinant DNA moleculecomprising a first nucleotide sequence coding for a target polypeptideand upstream to the first nucleotide sequence a second nucleotidesequence coding for an FKBP chaperone, wherein the FKBP chaperoneprovides for a functional chaperone activity, and wherein the FKBPchaperone allows for an increased cytosolic expression and in vitrorefolding of the fusion polypeptide; expressing the fusion polypeptide;obtaining the fusion polypeptide in the cytosol; solubilizing the fusionpolypeptide; and purifying the fusion polypeptide.